Figure 4

Decitabine-induced PKD1 reexpression in primary tumors. (A) Treatment schedule and timeline. MDA-MB-231 control and protein kinase D1 short hairpin RNA (PKD1 shRNA) cells (shRNA sequences 1 and 2) expressing luciferase were injected into the mammary fat pads of female nonobese diabetic severe combined immunodeficiency (NOD scid) mice. After establishment of primary tumors (days 1 to 14), mice were treated with 5 mg/kg decitabine or a saline solution injected intraperitoneally. Treatments were performed every other day over a period of 10 days. This was followed by a 10-day recovery phase. Overall, three cycles of treatment occurred, as shown in the timeline. (B) Analysis of PKD1 expression and PKD activity in primary tumors from mice with orthotopically implanted MDA-MB-231 scrambled control shRNA (scr-shRNA) cells and two different PKD1 shRNA lines (PKD1 shRNA 1 and 2) after control or decitabine treatment. Formalin-fixed, paraffin-embedded tissue was either stained with hematoxylin and eosin or analyzed for PKD1 expression (anti-PKD1) and PKD activity (anti-pS738/pS742) antibodies. Representative pictures of primary tumors are shown. (C) The number of PKD1-positive cells was calculated using the Aperio Positive Pixel Count algorithm in ImageScope viewing software. P values were calculated using Student’s t-test in GraphPad Prism version 5 software. *P ≤ 0.005, indicating statistical significance compared to saline-treated scr-shRNA cells. **P ≤ 0.005, indicating statistical significance compared to decitabine-treated scr-shRNA cells.